skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors

; ;  [1]
  1. (Toledo)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Biol. Chem.; Journal Volume: 289; Journal Issue: (36) ; 09, 2014
Country of Publication:
United States

Citation Formats

Favrot, Lorenza, Lajiness, Daniel H., and Ronning, Donald R.. Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors. United States: N. p., 2015. Web. doi:10.1074/jbc.M114.582445.
Favrot, Lorenza, Lajiness, Daniel H., & Ronning, Donald R.. Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors. United States. doi:10.1074/jbc.M114.582445.
Favrot, Lorenza, Lajiness, Daniel H., and Ronning, Donald R.. 2015. "Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors". United States. doi:10.1074/jbc.M114.582445.
title = {Inactivation of the Mycobacterium tuberculosis Antigen 85 Complex by Covalent, Allosteric Inhibitors},
author = {Favrot, Lorenza and Lajiness, Daniel H. and Ronning, Donald R.},
abstractNote = {},
doi = {10.1074/jbc.M114.582445},
journal = {J. Biol. Chem.},
number = (36) ; 09, 2014,
volume = 289,
place = {United States},
year = 2015,
month = 2
  • Previous studies identified ebselen as a potent in vitro and in vivo inhibitor of the Mycobacterium tuberculosis ( Mtb) antigen 85 (Ag85) complex, comprising three homologous enzymes required for the biosynthesis of the mycobacterial cell wall. In this study, the Mtb Ag85C enzyme was cocrystallized with azido and adamantyl ebselen derivatives, resulting in two crystallographic structures of 2.01 and 1.30 Å resolution, respectively. Both structures displayed the anticipated covalent modification of the solvent accessible, noncatalytic Cys209 residue forming a selenenylsulfide bond. Continuous difference density for both thiol modifiers allowed for the assessment of interactions that influence ebselen binding and inhibitormore » orientation that were unobserved in previous Ag85C ebselen structures. The k inact/ K I values for ebselen, adamantyl ebselen, and azido ebselen support the importance of observed constructive chemical interactions with Arg239 for increased in vitro efficacy toward Ag85C. To better understand the in vitro kinetic properties of these ebselen derivatives, the energetics of specific protein–inhibitor interactions and relative reaction free energies were calculated for ebselen and both derivatives using density functional theory. These studies further support the different in vitro properties of ebselen and two select ebselen derivatives from our previously published ebselen library with respect to kinetics and protein–inhibitor interactions. In both structures, the α9 helix was displaced farther from the enzyme active site than the previous Ag85C ebselen structure, resulting in the restructuring of a connecting loop and imparting a conformational change to residues believed to play a role in substrate binding specific to Ag85C. These notable structural changes directly affect protein stability, reducing the overall melting temperature by up to 14.5 °C, resulting in the unfolding of protein at physiological temperatures. Additionally, this structural rearrangement due to covalent allosteric modification creates a sizable solvent network that encompasses the active site and extends to the modified Cys209 residue. In all, this study outlines factors that influence enzyme inhibition by ebselen and its derivatives while further highlighting the effects of the covalent modification of Cys209 by said inhibitors on the structure and stability of Ag85C. Moreover, the results suggest a strategy for developing new classes of Ag85 inhibitors with increased specificity and potency.« less
  • GlgE is a bacterial maltosyltransferase that catalyzes the elongation of a cytosolic, branched α-glucan. In Mycobacterium tuberculosis (M. tb), inactivation of GlgE (Mtb GlgE) results in the rapid death of the organism due to a toxic accumulation of the maltosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. In this study, the crystal structures of the Mtb GlgE in a binary complex with maltose and a ternary complex with maltose and a maltosyl-acceptor molecule, maltohexaose, were solved to 3.3 Å and 4.0 Å, respectively. The maltohexaose structure reveals a dominant site for α-glucan binding. Tomore » obtain more detailed interactions between first generation, non-covalent inhibitors and GlgE, a variant Streptomyces coelicolor GlgEI (Sco GlgEI-V279S) was made to better emulate the Mtb GlgE M1P binding site. The structure of Sco GlgEI-V279S complexed with α-maltose-C-phosphonate (MCP), a non-hydrolyzable substrate analogue, was solved to 1.9 Å resolution, and the structure of Sco GlgEI-V279S complexed with 2,5-dideoxy-3-O-α-D-glucopyranosyl-2,5-imino-D-mannitol (DDGIM), an oxocarbenium mimic, was solved to 2.5 Å resolution. These structures detail important interactions that contribute to the inhibitory activity of these compounds, and provide information on future designs that may be exploited to improve upon these first generation GlgE inhibitors.« less
  • Peptide-based 1,2-dicarbonyl compounds have emerged as potent inhibitors for serine proteases. Herein, we have designed and synthesized D-arabinose and D-trehalose-based esters, {alpha}-ketoesters and {alpha}-ketoamides, and evaluated their inhibitory activity against Mycobacterium tuberculosis (Mtb) antigen 85C (ag85C), an acyltransferase in the serine hydrolase superfamily. In addition the compounds were evaluated for the ability to inhibit the growth of Mycobacterium smegmatis ATCC 14468, a non-pathogenic surrogate for Mtb. Among the synthetic analogs evaluated only the methyl ester1 derived from D-arabinose was found to inhibit the acyltransferase activity of ag85C (IC{sub 50} = 25 mM). Based on this weak inhibitory activity it wasmore » not surprising that none of the compounds inhibits the growth of M. smegmatis. In spite of the weak inhibitory activity of 1, X-ray crystallography on crystals of ag85C soaked with 1 suggested the formation of a covalent ester adduct between 1 and the Ser124 side chain hydroxyl moiety found within the catalytic site of ag85C; however, some of the active site electron density appears to result from bound glycerol. The lack of activity associated with the {alpha}-ketoester and {alpha}-ketoamide derivatives of D-trehalose may be the result of intramolecular cyclization of the {alpha}-keto moiety with the nearby C-4/4' hydroxyls leading to the formation of stable bicyclo-ester and amide derivatives.« less
  • The genome of Mycobacterium tuberculosis (TB) contains a gene that encodes a highly active {beta}-lactamase, BlaC, that imparts TB with resistance to {beta}-lactam chemotherapy. The structure of covalent BlaC-{beta}-lactam complexes suggests that active site residues K73 and E166 are essential for acylation and deacylation, respectively. We have prepared the K73A and E166A mutant forms of BlaC and have determined the structures of the Michaelis complex of cefamandole and the covalently bound acyl intermediate of cefamandole at resolutions of 1.2 and 2.0 {angstrom}, respectively. These structures provide insight into the details of the catalytic mechanism.